A small molecule inhibitor of inducible heat shock protein 70.

1Department of Genetics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6145, USA.

Abstract

The multifunctional, stress-inducible molecular chaperone HSP70 has important roles in aiding protein folding and maintaining protein homeostasis. HSP70 expression is elevated in many cancers, contributing to tumor cell survival and resistance to therapy. We have determined that a small molecule called 2-phenylethynesulfonamide (PES) interacts selectively with HSP70 and leads to a disruption of the association between HSP70 and several of its cochaperones and substrate proteins. Treatment of cultured tumor cells with PES promotes cell death that is associated with protein aggregation, impaired autophagy, and inhibition of lysosomal function. Moreover, this small molecule is able to suppress tumor development and enhance survival in a mouse model of Myc-induced lymphomagenesis. The data demonstrate that PES disrupts actions of HSP70 in multiple cell signaling pathways, offering an opportunity to better understand the diverse functions of this molecular chaperone and also to aid in the development of new cancer therapies.

(A) Whole cell extracts (WCE) prepared from A875 and BX-U2OS cells, treated with Biotin or Biotin-PES, were captured using NeutrAvidin agarose resins. HSP70-family proteins were identified as the major product in the excised band of ~ 70 kDa shown in both Coomassie gels. HSP70 and HSC70 peptide sequences are shown in .(B and C) WCE were prepared from the cell lines indicated following 24 h treatment with 20μM biotin or B-PES and examined for the expression of proteins indicated (left panel of B and C) by western blot analysis; note that different exposure times were used to visualize these proteins. B-PES-containing complexes were captured by NeutrAvidin Resins, and eluted following 100 mM DTT treatment. Immunoprecipitation-western blot (IP-WB) analysis using the indicated antibodies reveals interaction of B-PES with endogenous HSP70, but there is no detectable interaction with endogenous BAK, BCL-xL, GRP78, p53, HSC70, or HSP90 even after longer exposure times.(D) In vitro evidence for an interaction between B-PES and HSP70. Full-length human HSP70 and human HSP90 proteins were in vitro translated in the presence of 35S-methionine, mixed with B-PES coupled to NeutrAvidin resins, and eluted using 100 mM DTT. The resulting DTT eluates were separated by polyacrylamide gel electrophoresis (SDS-PAGE) and visualized by autoradiography.(E) B-PES interacts with the C-terminal region of HSP70. H1299 cells were transfected with the indicated hemagglutinin (HA)-tagged constructs and exposed to B-PES. B-PES containing complexes were captured by NeutrAvidin Resins, eluted following 100 mM DTT treatment, and immunblotted with anti-HA antibody following SDS-PAGE.

(A) WCE from H1299 lung carcinoma cells, treated either with 20μM Biotin or B-PES for 24 h, were immunoprecipitated (IP) using anti-HSP70 antibody. Western blots assessed the relative abundance of the proteins indicated (left), and co-immunoprecipitation-western (IP-WB) analysis revealed a reduced degree of interaction between HSP70 and HSP40, CHIP, BAG-1M, and APAF1 in B-PES-treated cells (right).(B) IP-WB analyses of WCE from PES-treated U2OS osteosarcoma cells reveal a lower abundance of HSP70 complexes containing BAG-1M or APAF1.(C) WCE were prepared from A875 melanoma cells that were either untreated or pretreated with PES (20μM) for 1 h, followed by the addition of 50μM cisplatin for 8 h. Note evidence of caspase cleavage in cisplatin-treated cells, but not in the presence of PES. IP-WB analysis reveals the presence of p53/BAK and p53/HSP70 complexes in cisplatin-treated cells (right) that are reduced following exposure to PES.(D) (left) H1299 cells were transfected with a NF-κB-dependent luciferase reporter. 24 h later, cells were either pretreated with DMSO or the indicated amount of PES for 1 h, followed by the addition of 20 ng/ml of TNFα for 5 h, as specified. Each graphical representation indicates the mean ± SD of at least three independent cultures relative to control (DMSO-treated) cells. (right) H1299 cells were treated with 20μM PES for 5.7 h, followed by treatment with 10 ng/ml TNF for 20 min. WCE were immunoblotted for the proteins indicated.

(A) MTT assays of A875 cells treated with DMSO, 20μM PES, or 50μM cisplatin for 24 h. Each graphical representation indicates the mean ± SD of at least three independent cultures relative to control (DMSO-treated) cells.(B) (Top) WCE were prepared from U2OS osteosarcoma cells that were either untreated or pretreated with Z-VAD-FMK (20μM) for 1 h, followed by the addition of 5μM camptothecin (Camp), 16μg/ml Cisplatin (Cis), or 20μM PES for 6 h. Note evidence of caspase cleavage in cisplatin- or camptothecin-treated cells, but not in the presence of PES or Z-VAD-FMK. (Bottom) The indicated cell lines were either untreated or pretreated with Z-VAD-FMK (20μM) for 1 h, followed by the addition of DMSO or 20μM PES for 24 h. Cell viability was determined by MTT assays. Results shown are the mean of at least three independent experiments.(C) The indicated cell lines were treated with the indicated concentrations of PES for either 24 h (top) or for 48 h (middle and bottom). Representative MTT assays indicate cell viability in human cell lines, including non-transformed human WI38 fibroblasts, as well as several tumor cell lines with wt p53 (U2OS, BX-U2OS, MCF7, CaPan2), or with mutant/deleted p53 (SKBR3, MDA468, MDA231, CaPan1, MiaPaCa, and Panc1). Four independent cultures were assayed for each treatment and DMSO treated cells were used as internal controls. Values shown are normalized to the viability of the control (DMSO-treated) cells. Error bars represent standard deviation (SD).

(A) Western blot (WB) analysis reveals increased appearance of processed LC3-II in the indicated cells following 20μM PES treatment for the indicated times.(B-H) Electron micrographs of H1299 cells with or without PES treatment (20μM) for 7 or 24 h. (D) Double membrane autophagic vacuoles (AV), and vacuoles within vacuoles are evident. (E and F) AVs of different sizes are evident, some containing recognizable cytoplasmic content. (G and H) Large AVs containing partially digested cytoplasmic material as well as amorphous, membranous, aggregated, or granular masses are shown.(I) The average area of autophagic vacuoles (AV) calculated with ImageJ software per cell is indicated.

(A) Decreased degradation of long-lived proteins in U2OS cells treated with PES (20μM) for the indicated time (black line), compared to vehicle treated cells (gray line). Data are reported in percent of protein degraded at each time point, and are the averaged data from two independent experiments done in duplicate; error bars represent standard deviation. The data were consistent in H1299 cells (data not shown).(B) Western blot (WB) analysis indicating altered processing of cathepsin L from the larger precursor form to the smaller mature form in the indicated cells following 20μM PES treatment for 24 h.(C) IP-WB analyses of WCE from untreated or PES-treated A875 melanoma cells (left) and MiaPaCa pancreatic cells (right) reveal a lower abundance of HSP70 complexes containing LAMP2 following 20μM PES exposure for 24 h (lower panels). The expression patterns of LAMP2 before and after 20μM PES exposure for 24 h are shown on the top panel.

(A and B) Western blot (WB) analysis showing increased appearance of processed LC3-II and p62/SQSTM1 oligomerization in the indicated cells following 20μM PES treatment for the indicated time.(C) MCF7 cells were treated with indicated amount of PES for 24 h, and examined for the indicated proteins.(D) Immunostaining for p62/SQSTM1 in BX-U2OS cells, either before or following 20μM PES treatment for 24 h. Note the appearance of p62/SQSTM1 punctae and inclusion bodies.(E) IP-WB analyses of WCE from vehicle or PES-treated MiaPaCa pancreatic cells (top panel) and A875 melanoma cells (lower panel). Note that LC3 binds to both the monomeric and oligomeric forms of p62/SQSTM1 following PES-exposure.(F) WI38 or H1299 cells were transfected with a negative shRNA or with HSP70 shRNAs, and examined for the indicated proteins.(G) WI38 cells were transfected with a negative shRNA or with HSP70 shRNAs. After 72 h, the cells were either untreated or treated with 10μM PES for 24 h, and examined for the indicated proteins.(H) H1299 cells were transfected with a negative shRNA or with HSP70 shRNAs. After 48h, the cells were either treated with DMSO or 20μM PES for 24 h. Each graphical representation indicates the mean ± SD of at least three independent cultures relative to control cells transfected with a negative shRNA and treated with DMSO.(I) MCF7 cells were treated either with 20μM PES or 100 nM Velcade for 24 h before harvesting cells in NP40-containing lysis buffer. Lysates were centrifuged to separate the clarified lysate (detergent-soluble) and NP40-insoluble (detergent-insoluble) fractions and assayed by western blot for the proteins indicated.(J) BX-U2OS cells were treated with 20μM PES, 50μM chloroquine (CQ), or 15 nM 17-AAG for 24 h. Cells were harvested in 1% NP40-containing lysis buffer, fractionated into detergent-soluble and detergent-insoluble preparations, and assayed by western blot for the proteins indicated.(K) A875 cells were either pre-treated with DMSO or 20μM PES for 1 h prior to the addition 50μg/ml of cycloheximide (CHX) for 5 h. Note the marked reduction in PES-induced p62/SQSTM1 oligomerization in the insoluble fraction, the significant inhibition of LC3-II processing following PES and CHX co-treatment, and the obvious loss of cathepsin L expression in the presence of CHX.

Beginning at 8 weeks of age, Eμ-Myc transgenic mice were treated either with vehicle or 40 mg/kg PES intra-peritoneally every 5 days for a total of 30 days (n = 13 for each group). The black arrow indicates the first day of treatment (day 56), and the open arrow indicates the final day of treatment (day 86). Note that PES-administration increased the overall mean survival time from about 80 days in vehicle treated animals to > 140 days in PES-treated cohorts.